Shen G, Fu X, Zhou B, Yin J, Zhong C, Chen J, Hou J
Duck Hepatitis B Virus DNA Copy Numbers in Isolated Hepatocyte Nuclei Vary Dramatically and Decline During Entecavir Therapy.
Antiviral Therapy. 2013 Jun 14;
BACKGROUND: We aimed to develop a quantitative assay to measure duck hepatitis B virus (DHBV) DNA in single hepatocyte nuclei from DHBV-infected animals and to observe intranuclear DHBV DNA kinetics undergoing entecavir (ETV) therapy. METHODS: DHBV DNA in isolated nuclei was amplified by quantitative real-time polymerase chain reaction (PCR). Liver tissues from chronically infected ducks with or without ETV treatment were assessed. Cell cycle phases were defined with flow cytometry in single nuclei. RESULTS: We successfully established a quantitative assay to measure intranuclear DHBV DNA in single nuclei with high specificity, sensitivity and acceptable interassay variations. The intranuclear viral DNA copy numbers varied dramatically (2-204 copies/nuclei) in 11 ducks with active viral replication. Average intranuclear DHBV DNA copies from individual animals (7.57-57.67/nuclei) significantly correlated with total intranuclear (rs = 0.955, P < 0.001) and serum (rs = 0.745, P = 0.008) viral DNA levels. The median intranuclear DHBV DNA copies in virus-positive nuclei were greater in G0/1 than those in G2/M and S phases (P < 0.001). Median intranuclear viral DNA copies in virus-positive nuclei decreased from 21 to 6 (P < 0.001) under 14-19 weeks of ETV therapy. However, subsequently, no further reductions were not achieved in 4 animals after extended 16 week treatment (6 vs. 11, P = 0.034). CONCLUSIONS: Intranuclear DHBV DNA levels varied significantly, which could be partially attributed to impacts from cell cycle phases, and could be decreased by ETV therapy.